Exam Three

Whole Exome Sequencing

sequencing only genes with exons

2% of genome

cheaper and faster

Filtering Scheme

keep: variants in/near exons, rare variants not in most people, drastic variants, compound heterozygous variants, homozygous recessive variants

consider which genes are expressed in affected tissues and which genes are mutated in other affected individuals

Postaxial Acrofacial Dysostosis (POAD)

results from an autosomal recessive mutation

affected individuals have a small jaw and missing digits

results from a mutation in four possible genes (DHODH gene likely)

Loss of Function Mutation

mutation resulting in no expression or protein does not work

severity depends on percent of residual function

Gain of Function Mutation

mutation resulting in enhanced function or altered expression

Overexpression

gain of function mutation resulting in too much protein

caused by duplications

Enhanced Protein Function

gain of function mutation resulting in a protein performing its function better

Heterochronic Expression

gain of function mutation that results in gene expression at the wrong time

example: hereditary persistence of fetal hemoglobin

Ectopic Expression

gain of function mutation resulting in gene expression in the wrong place

New Function Mutation

mutation resulting in the protein doing something that it does not usually do

may not affect normal function

Allelic Heterogeneity

many alleles for one gene (more than two)

Null Allele

gene has no function

Locus Heterogeneity

many genes cause one disease

Hemoglobinopathies

class of diseases that affect hemoglobin

most common single gene disorder

5% of the world are carriers

Hemoglobin (Hb)

carry oxygen in red blood cells

tetramer (four units)

α-Globin

globin with fetal and adult expression

constitutive expression

γ-Globin

globin with only fetal expression

repressed after birth

β-Globin

globin with only adult expression

Sickle Cell

homozygous recessive disorder causing red blood cells to change shape

GAG to GTG causing Glu to Val

disrupts cell membrane causing hemolysis and anemia

cells do not fit through capillaries, blocking circulation

Sickle Cell Trait

heterozygous for β-globin gene

may have attacks when oxygen is low

BCL11A

gene codes for a protein that represses  γ-globin after birth

mutating this gene used as therapy for sickle cell

Housekeeping Genes

ubiquitous expression

mutations may not affect all tissues because the gene may not be expressed at the same levels or a similar gene may rescue it in some tissues

Specialty Genes

tissue-specific expression for unique function in that tissue

mutations may not cause issues in the specific tissue

Phenylketonuria (PKU)

results from a PAH enzyme mutation

phe accumulation leads to brain damage

treatment is lowering phe intake by avoiding foods like meat, eggs, and milk

Variant PKU

greater than 1% PAH activity (residual activity)

less severe

Familial Hypercholesterolemia

results from mutation in LDLR or three other genes

increased lipids in the blood

PCSK9 Protease

degrades LDLR

gain of function mutation degrades too much LDLR

loss of function mutation does not degrade LDLR and reduces heart disease risk

Cystic Fibrosis (CF)

mutation in chloride transport regulator

results from mutations to CFTR gene

affects lungs, endocrine glands, digestion, sweat (salty)

CFTR Gene

codes for large membrane protein to transport chloride

190 kb with 27 exons

mutations cause CF

SCNN1 Gene

codes for sodium channel

when mutated, phenotype mimics CF

Pleiotropic

one gene affects many tissues

Clinical Heterogeneity

different alleles have different phenotypes

Duchene Muscular Dystrophy (DMD)

results from mutation in dystrophin gene

X-linked recessive disorder

muscle deterioration, muscle weakness, heart and lung complications

no therapy, treat symptoms

Dystrophin Gene

large gene (2,300 kb with 79 exons) that codes for a muscle protein

has seven tissue specific promoters

causes DMD when mutated

MYB

modifier gene that represses  γ-globin expression in adults

Trisomy 13

having three copies of chromosome 13

increases miRNAs that are inhibitors of MYB increasing γ-globin expression

Unstable Hemoglobins

Phe-42-Ser mutation in β-globin making it unstable causing it to unfold

alpha tetramer

damages red blood cells and causes hemolysis

Thalassemia

most common single gene disease

heterozygous advantage for malaria

decrease in synthesis or stability of globins

α-Thalassemia

decrease in α-globin

fetal and adult disease

do not release oxygen (hypoxia)

 β-Thalassemia

decrease in β-globin and excess α-globin precipitate

red blood cell damage

only in adults

mostly point mutations

Mitochondrial Chromosomes

circular with 37 genes for oxidative phosphorylation

make proteins, rRNAs, and tRNAs

work with 74 nuclear genes

maternal inheritance

Replicative Segregation

mtDNA replicates and sorts randomly to new mitochondria (mitochondria with less DNA makes less ATP)

mitochondria replicates and sorts randomly into new cells

Mitochondrial Disease

results from mutations in mtDNA or mutations in nuclear DNA that is involved in oxidative phosphorylation

affects high metabolism tissues

Nuclear Transfer

treatment for women with mitochondrial diseases who want to have children

mother's nucleus is put into a donor egg with no nucleus but healthy mitochondria

approved in UK and being reviewed in US

Challenges with Treatment of Genetic Diseases

unknown gene or unknown pathogenesis

fetal diseases are difficult to diagnose

null alleles difficult to treat

dominant alleles difficult to treat (similar to wild type)

initial treatment success can still have long term defects

organ targeting leaves other affected organs

genetic heterogeneity

Substrate Reduction

strategy to manipulate metabolism by decreasing the amount of substance available

decreasing Phe intake in PKU patients

Replacement

strategy to manipulate metabolism in response to a decrease in metabolite, cofactor, or hormone

treat decreased thyroxin from congenial hypothyroidism with thyroxine supplements

Diversion

strategy to manipulate metabolism by enhancing an alternate pathway to decrease a harmful metabolite

benzoate to decrease ammonia in patients with mutation affecting urea cycle

Enzyme Inhibition

strategy to manipulate metabolism by stopping an enzyme from working

inhibit PCSK9 enzyme that breaks down LDLR allowing for more cholesterol uptake

Receptor Antagomism

strategy to manipulate metabolism where a substance binds to a receptor, decreasing the affect of a substrate

losartan to lower TGF-B expression in patients with Marfan syndrome

Depletion

strategy to manipulate metabolism that removes a harmful metabolite

apheresis procedure to remove LDL from blood of patient before returning it

Strategies for Metabolism Manipulation as Treatment for Genetic Diseases

six strategies

substrate reduction, replacement, diversion, enzyme inhibition, receptor antagonism, and depletion

Strategies for Treatment at the Protein Level

codon skipping, chaperones, potentiator, enhance enzyme function, and protein replacement

Chaperones

help proteins fold

can be used to treat diseases that result from improper protein folding

Potentiators

increase the function of membrane proteins

can be used to treat diseases that result from decreased protein function

Cofactors

vitamins or other substances that increase enzyme function

can be used to treat diseases that result from decreased enzyme function

cannot treat diseases that result from null alleles

Protein Replacement

adding in the needed protein

can be direct (just giving the protein)

can be indirect (transplants and gene therapy)

Strategies to Modulate Gene Expression

increase gene expression and increase expression of another (modifier) gene

Gene Editing

used to change a gene

can use CRISPR/Cas

CRISPR/Cas

used for gene editing

nuclease uses gRNA to target a gene and cut it

cells then undergo homologous recombination repair

Stem Cells

multipotent (differentiate into many cell types) and self-renew

can be used to treat genetic diseases

Hemopoietic Stem Cells (HBCs)

make all new blood cells

can be used to treat blood cell diseases like SCID, sickle cell, and β-thalassemia

Induced Pluripotent Stem Cells (iPSCs)

taking a person’s own already differentiated cell and turning it into a stem cell

gives a personalized immune match

Organ Transplant Limitations

immune match and chance of rejection

increased infection from immunosuppressant drugs

low supply

Gene Therapy

adding a wild-type gene using a viral vector or plasmid

number of normal cells needed is important

Viral Vector

used in gene therapy to add a wild-type gene into the chromosome

problem with insertional mutagenesis (breaking up a gene during insertion)

should not be able to replicate, not be toxic, cause no allergic reactions, and have a low number of integrations

Plasmid

could be used for gene therapy to introduce wild-type gene

stays in cytoplasm and does not insert into chromosome

Diseases Treated Using Gene Therapy

eye degeneration, SCID, hemophilia B, and β-Thalassemia

most current trial focus on cancer and some monogenic diseases

Reasons Eye Diseases are Good Candidates for Gene Therapy Treatments and Trials

not a vital organ

easy to access

easy to assess